The vir locus of B. pertussis is responsible for the coordinate regulation of many of the virulence associated factors. Using TnphoA mutagenesis several new vir activated genes (vags) have been identified. One mutant strain, SK8, is defective in a 95kDa outer membrane protein. This strain has been shown to be unable to cause lymphocytosis or to persist in the mouse aerosol model of pertussis. The wild type gene has been isolated from a cosmid library and is being sequenced. Preliminary evidence suggests that this gene may be part of an operon and we are investigating this possibility. A monoclonal antibody, 8E7, has been purified for use in an affinity column to purify the protein. Strain SK34, which has a TnphoA insertion in vag-34, lacks 4 outer membrane proteins of 92, 90, 60 and 30 kDa. Vag-34 encodes a 60kDa product. The C terminal portion of this protein is very similar to that of the pertactin precursor. the N terminal 36kDa portion of the molecule contains a RGD site and is very proline rick (15%). When vag-34 DNA encoding the N terminal portion of the protein is placed in a SK34 background the 60 and 90kDa bands are regenerated. We are currently investigating whether the C terminal portion generates the 30kDa band and whether this portion of the molecule is important for secretion, targeting or anchoring of the protein in the membrane. In the mouse aerosol model SK34 appears less able to persist in the tracheas of mice than the parent strain. this behavior is similar to strains defective in the known adhesion FHA, thus we are investigating whether vag-34 may have a role in adhesion. Strain SK49 is defective in a porin like protein. This regulated porin is similar to the major constitutive porin but is expressed at much lower levels. Although this protein is regulated by the vir regulon preliminary evidence suggests that is may also be autoregulated; this possibility is under investigation.